MicroRNAs (miRs) are a new class of small non-coding RNAs that may regulate up to 30% of human genes. miRs are involved in a myriad of cellular processes: proliferation, differentiation, apoptosis and responses to stress, and were shown to be involved in normal cholangiocyte physiology as well as in cholangiocarcinomas (CCA). CCAs are cancers arising from the biliary tract. They are the 2nd most common primary hepatobiliary cancers in the United States, and their incidence is increasing globally. The survival of patients with CCA is dismal, usually measured in months. Our preliminary studies in cholangiocytes identified one hundred and thirty two miR species with differential expression in cholangiocytes vs. neoplastic cholangiocytes. We hypothesize that among these miRs, there are several miR species that have an etiologic role in neoplasia formation and homeostasis. Furthermore, we hypothesize that these miRs have important role in the normal cholangiocyte homeostasis, regulating cell cycle progression, proliferation and/or apoptosis. In the current application, we propose a step- wise approach to identify miRs with phenotypic impact, followed by careful characterization of their function. Specifically, we propose to: 1. Identify 4 miRs that have a phenotypic impact on cholangiocytes in vitro (Aim 1). We will select miR species that have similar phenotypic impact on human (Aim 1a) and murine (Aim 1b) cell lines. The reason for this restriction is that we plan on using the SMAD4/PTEN murine model of cholangiocarcinoma for our in vivo studies. Next, we propose to 2. Further validate the in vitro phenotypic action of the candidate miRs through in vivo studies (Aim 2). By applying a step-wise algorithm, we plan on verifying the miR action in a xenograft model (Aim 2a) and then to further study the miRs with phenotypic impact in a liver-specific SMAD4/PTEN-deleted murine model of CCA. At the conclusion of Aims 1 and 2, we will have identified 4 miR species with phenotypic impact (cell cycle, proliferation, apoptosis) on cholangiocytes in vitro and in vivo. These miR species are very likely to be involved in normal cholangiocyte cellular processes as well as altered as cholangiocytes become neoplastic. Finally, we propose to 3. Identify mechanism of action for miR species identified in Aim 1 and confirmed in Aim 2 (Aim 3). We will identify mRNA and protein targets for these miRs as well as explore the pathways connecting these miRs to their mRNA targets. I believe that through funding this application and execution of the proposed experiments, several goals will be met: 1. my training in molecular biology will be perfected with the final goal of contributing to my independence as principal investigator; 2. the current understanding of the normal physiology of cholangiocytes as well as the neoplastic transformation will be advanced. Ultimately, I am hopeful that through the findings of this project, we will make significant progress towards understanding how and why inflammatory conditions of the biliary tree, such as Primary Sclerosing Cholangitis, progress to neoplasia. PUBLIC HEALTH RELEVANCE: Our preliminary studies in cholangiocytes identified 4 microRNA species with profound effects on cell growth, cell cycle progression and apoptosis in vitro. In the current study, we propose to further dissect the effects of these microRNAs on cholangiocytes in vitro, as well as testing their effects in vivo. Finally, we aim at uncovering the mechanistic links between these microRNAs and known normal or neoplastic pathways in cholangiocytes, with the final purpose of better understanding the normal physiology of cholangiocytes as well as the neoplastic transformation.